Thesis (M.A.)--University of Calgary, 2005. / Includes bibliographical references (p. 110-117)

In part ONE of this thesis, including the most up to date information on SARS virology, disease transmission, pathogenesis and laboratory diagnosis will be summarized and presented, including the results of many studies in which I have participated (these references will be underlined as they appear in text). This of course summarizes knowledge that is now known in 2006 but was largely unknown during the initial outbreak. In part TWO, six original clinical studies performed at PWH will be presented: study (1) describes the clinical manifestations and severity of SARS, and its potential to cause major hospital outbreaks; (2) demonstrates the importance of epidemiological linkage in diagnosing SARS; (3) reports the clinical outcomes of a stepwise treatment protocol, which includes the use of corticosteroid therapy as an immunomodulant; (4) demonstrates that corticosteroid therapy can retard viral clearance, and should be used judiciously; (5) demonstrates that a more robust humoral response is associated with severe SARS, thus indicating that passive immunity treatment strategies seem only suitable either during early illness or as prophylaxis; and (6) shows that SARS has few early discriminating laboratory features compared to other causes of community-acquired pneumonia, thus a high index of suspicion is needed to recognize this infection in the absence of worldwide transmission. A thorough review of the relevant published material will be included in the discussion section of each study. / Severe Acute Respiratory Syndrome (SARS) is an emerging infectious disease caused by a novel coronavirus. It caused a global outbreak in 2003, resulting in more than 8000 infections, 700 deaths, and major social and economic disruption. In the initial phase of the SARS outbreak, the medical profession had no knowledge regarding the responsible pathogen, nor the clinical manifestations of SARS and the course of illness. There was no reliable diagnostic tool and no known effective therapy. But for the first time in medical history, we witnessed the rapid accumulation of knowledge on a disease as it evolved, which in turn assisted its management and control. / Since conducting randomized-controlled trials during the 2003 crisis was almost impossible, most of the presented studies are either descriptive or case-controlled in design. However, these studies have laid foundations for recent and future research into the clinical diagnosis and management of SARS. Moreover, the construction of the SARS clinical database has contributed to the work of other investigators, which has resulted in over thirty-six publications. It is my hope that these research endeavors can contribute to the understanding of this emerging, deadly disease. / Lee Lai Shun, Nelson. / "April 2006." / Source: Dissertation Abstracts International, Volume: 69-01, Section: B, page: 0205. / Thesis (M.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 264-292). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

published_or_final_version / abstract / Microbiology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Pathology / Master / Master of Philosophy

published_or_final_version / abstract / Biological Sciences / Master / Master of Philosophy

published_or_final_version / abstract / Chemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

Poon Wing Ming Jodie. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 207-233). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / 論文摘要 --- p.iv / Abbreviations --- p.v / List of Figures --- p.x / List of Tables --- p.xiii / Contents --- p.xiv / Chapter CHAPTER ONE --- INTRODUCTION --- p.1 / Chapter 1.1. --- Severe Acute Respiratory Syndrome (SARS) --- p.1 / Chapter 1.1.1. --- Background of SARS --- p.1 / Chapter 1.1.2. --- Etiology and pathology of SARS --- p.3 / Chapter 1.1.3. --- Genome organization and expression of SARS-CoV --- p.5 / Chapter 1.1.4. --- Current molecular advances of SARS-CoV --- p.13 / Chapter 1.1.5. --- Current research advances on SARS-CoV nucleocapsid --- p.18 / Chapter 1.1.6. --- Current diagnostic assays of SARS-CoV infection --- p.23 / Chapter 1.1.7. --- Current treatment --- p.25 / Chapter 1.1.8. --- Vaccine development --- p.27 / Chapter 1.2. --- Aims of study --- p.30 / Chapter CHAPTER TWO --- MATERIALS AND METHODS --- p.33 / Chapter 2.1. --- Subcellular localization study of the SARS-CoV nucleocapsid protein --- p.33 / Chapter 2.1.1. --- "Cloning of SARS-CoV nucleocapsid cDNA into the green fluorescence protein (GFP) mammalian expression vector, pEGFP-C1" --- p.33 / Chapter 2.1.1.1. --- Amplification of SARS-CoV nucleocapsid gene by polymerase chain reaction (PCR) --- p.33 / Chapter 2.1.1.2. --- Purification of PCR products --- p.35 / Chapter 2.1.1.3. --- Restriction digestion of purified PCR products and the circular pEGFP-C 1 vector --- p.36 / Chapter 2.1.1.4. --- Ligation --- p.36 / Chapter 2.1.1.5. --- Preparation of chemically competent bacterial cell E.coli strain DH5a for transformation --- p.37 / Chapter 2.1.1.6. --- Transformation of ligation product into chemically competent bacterial cells --- p.38 / Chapter 2.1.1.7. --- Small-scale preparation of bacterial plasmid DNA --- p.39 / Chapter 2.1.1.8. --- Screening for recombinant clones --- p.40 / Chapter 2.1.1.9. --- DNA sequencing of cloned plasmid DNA --- p.41 / Chapter 2.1.1.10. --- Midi-scale preparation of recombinant plasmid DNA --- p.42 / Chapter 2.1.2. --- Cell culture --- p.44 / Chapter 2.1.2.1. --- Sub-culture of VeroE6 and HepG2 cell lines --- p.44 / Chapter 2.1.2.2. --- Transient transfection of GFP fusion construct --- p.45 / Chapter 2.1.3. --- Epi-fluorescent microscopy --- p.46 / Chapter 2.2. --- Study on differential gene expression patterns upon SARS-CoV nucleocpasid induction by cDNA microarray analysis --- p.48 / Chapter 2.2.1. --- Cloning of SARS-CoV N gene into mammalian expression vector pCMV-Tagl --- p.48 / Chapter 2.2.2. --- Cell culture --- p.50 / Chapter 2.2.2.1. --- Sub-culture of VeroE6 cell line --- p.50 / Chapter 2.2.2.2. --- Transient transfection of pCMV-Tag1 -SAR-CoV N construct --- p.50 / Chapter 2.2.3. --- Total RNA isolation --- p.51 / Chapter 2.2.3.1. --- Total RNA isolation by RNeasy Mini Kit --- p.51 / Chapter 2.2.3.2. --- Checking of RNA integrity --- p.53 / Chapter 2.2.3.3. --- Checking of RNA purity --- p.54 / Chapter 2.2.3.4. --- Determinations of total RNA concentrations and precipitation --- p.54 / Chapter 2.2.4. --- cDNA microarray (done by Affymetrix Inc. as a customer service) --- p.55 / Chapter 2.2.4.1. --- Precipitation of RNA --- p.55 / Chapter 2.2.4.2. --- Quantification of RNA --- p.56 / Chapter 2.2.4.3. --- Synthesis of double-stranded cDNA from total RNA --- p.56 / Chapter (i) --- First stand cDNA synthesis --- p.56 / Chapter (ii) --- Second cDNA synthesis --- p.57 / Chapter 2.2.4.4. --- Clean-up of double stranded cDNA --- p.58 / Chapter (i) --- Phase lock gel-phenol/ chloroform extraction --- p.58 / Chapter (ii) --- Ethanol precipitation --- p.58 / Chapter 2.2.4.5. --- Synthesis of biotin-labeled cRNA --- p.59 / Chapter 2.2.4.6. --- Clean-up and quantification of in vitro transcription (IVP) products --- p.59 / Chapter (i) --- In vitro transcription clean-up --- p.59 / Chapter (ii) --- Ethanol precipitation --- p.60 / Chapter (iii) --- Quantitation of cRNA --- p.60 / Chapter (iv) --- Sample checking --- p.60 / Chapter 2.2.4.7. --- cRNA fragmentation for target preparation --- p.60 / Chapter 2.2.4.8. --- Eukaryotic target hybridization --- p.61 / Chapter 2.2.4.9. --- "Probe array washing, staining and scanning" --- p.62 / Chapter 2.2.5. --- Confirmation of results by RT-PCR --- p.62 / Chapter 2.2.5.1. --- First-strand cDNA synthesis --- p.62 / Chapter 2.2.5.2. --- RT-PCR of candidate gene --- p.63 / Chapter 2.3. --- In vitro RNA interference of SARS-CoV nucleocapsid --- p.66 / Chapter 2.3.1. --- siRNA target site selection --- p.66 / Chapter 2.3.2. --- Cloning of target siRNA sequences into pSilencer 3.1-H1 vector --- p.71 / Chapter 2.3.3. --- Cell culture --- p.72 / Chapter 2.2.3.1. --- Sub-culture ofVeroE6 cells --- p.72 / Chapter 2.3.3.2. --- Transient co-transfection --- p.72 / Chapter 2.3.4. --- Detection of SARS-CoV nucleocapsid mRNA expression level by RT-PCR --- p.73 / Chapter 2.3.4.1. --- Total RNA isolation by TRIzol reagent --- p.73 / Chapter 2.3.4.2. --- First-strand cDNA synthesis --- p.74 / Chapter 2.3.4.3. --- RT-PCR assays --- p.74 / Chapter 2.3.5. --- Detection of SARS-CoV nucleocapsid protein expression level by Western blotting --- p.75 / Chapter 2.3.5.1. --- Total protein extraction --- p.75 / Chapter 2.3.5.2. --- Protein quantification --- p.75 / Chapter 2.3.5.3. --- Protein separation by SDS-PAGE and Western blot --- p.76 / Chapter 2.3.5.4. --- Western blot analysis --- p.78 / Chapter 2.4. --- Human fgl2 prothrombinase promoter analyses --- p.80 / Chapter 2.4.1. --- Cloning of the full-length human fgl2 prothrombinase promoter construct into a promoterless mammalian expression vector-pGL3-Basic --- p.80 / Chapter 2.4.2. --- Cloning of SARS-CoV Membrane gene into the mammalian expression vector pCMV-Tagl --- p.82 / Chapter 2.4.3. --- Cell culture --- p.84 / Chapter 2.4.3.1. --- Sub-culture of HepG2 and VeroE6 cell lines --- p.84 / Chapter 2.4.3.2. --- "Transient co-transfection of the full-length human fgl2 prothrombinase promoter construct with the pCMV-Tagl empty vector, pCMV-Tagl-SARS-CoV M expression vector, or pCMV-Tag1 -SARS-CoV N expression vector" --- p.84 / Chapter 2.4.4. --- Dual-luciferase reporter assay --- p.85 / Chapter 2.4.5. --- Detection of fgl2 mRNA expression level under the induction of SARS-CoV nucleocapsid protein by RT-PCR --- p.86 / Chapter 2.4.5.1. --- Total RNA isolation by TRIzol reagent --- p.86 / Chapter 2.4.5.2. --- First strand cDNA synthesis --- p.86 / Chapter 2.4.5.3. --- RT-PCR of fgl2 gene --- p.87 / Chapter CHAPTER THREE --- RESULTS --- p.88 / Chapter 3.1. --- Computer analysis of SARS-CoV Nucleocapsid --- p.88 / Chapter 3.2. --- Subcellular localization of SARS-CoV nucleopcasid protein in VeroE6 cells and HepG2 cells --- p.102 / Chapter 3.3. --- cDNA microarray analysis on differential gene expression pattern upon the over-expression of SARS-CoV Nucleocapsid gene --- p.114 / Chapter 3.4. --- In vitro RNA Interference of SARS nucleocapsid --- p.129 / Chapter 3.5. --- Transactivation of fgl2 prothrombinase gene promoter by SARS-CoV nucleocapsid protein in HepG2 and VE6 cells --- p.138 / Chapter CHAPTER FOUR --- DISCUSSION --- p.155 / Chapter 4.1. --- "The EGFP-tagged SARS-CoV N protein was localized in the cytoplasm only in VE6 cells, but translocated into both cytoplasm and nucleus in HepG2 cellsin the epi-fluorescence microscopy study" --- p.155 / Chapter 4.2. --- cDNA microarray demonstrated alternations of mRNA transcript level on a number of genes belonging to various functional classes upon over expression of SARS-CoV nucleocapsid gene --- p.162 / Chapter 4.3. --- RNA interference demonstrated effective gene silencing of SARS-CoV nucleocapsid gene --- p.171 / Chapter 4.4. --- SASR-CoV nucleocapsid protein induced the promoter activity of the prothrombinase fibrinogen-like protein2/ fibroleukin (fgl2) gene --- p.191 / Chapter 4.5. --- Conclusion --- p.196 / Chapter 4.6. --- Future work --- p.198 / Appendices --- p.199 / References --- p.207

Thesis (M. Phil.)--University of Hong Kong, 2007. / Also available online.

Thesis (M. Phil.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.